T-cell receptor antagonist modifies cytokine secretion profile of naive CD4+ T cells and their differentiation into type-1 and type-2 helper T cells

A T-cell receptor (TCR) antagonist is an analog of a peptide ligand for TCR that inhibits T-cell responses to the original peptide. We investigated the effects of a TCR antagonist on cytokine secretion of naive CD4+ T cells and their differentiation into type-1 and type-2 helper T cells (Th1 and Th2) induced by stimulation with varying doses of an antigenic peptide. In the presence of a TCR antagonist peptide, proliferation of naive CD4+ T cells and antigen dose-dependent secretion of interferon-gamma, a typical Th1-type cytokine, by these cells was down-regulated. With respect to the secretion of interleukin-4 (IL-4), a typical Th2-type cytokine, the TCR antagonist raised the concentration of the antigenic peptide required to elicit maximal IL-4 production and, surprisingly, significantly increased the maximum level of IL-4 secretion. Similar effects induced by the TCR antagonist were observed on the Th1/Th2 differentiation of naive CD4+ T cells. These results clearly indicate that, for naive CD4+ T cells, a TCR antagonist has the potential to change the balance of Th1/Th2 cytokine secretion and even enhance Th2 responses.     

Modification of in vivo and in vitro T- and B-cell-mediated immune responses by the Pseudomonas aeruginosa quorum-sensing molecule N-(3-oxododecanoyl)-L-homoserine lactone

N-3-(oxododecanoyl)-L-homoserine lactone (OdDHL), a quorum-sensing molecule of Pseudomonas aeruginosa, plays an important role in the pathogenesis of the organism through its control of virulence factor expression. Several reports have suggested that OdDHL can also directly modulate host immune responses. However, the nature of the modulation is controversial, with different reports suggesting promotion of either humoral (Th2-mediated) or inflammatory (Th1-mediated) responses. This report describes a series of studies which demonstrate for the first time that in vivo administration of OdDHL can modulate the course of an antibody response, with an increase in ovalbumin (OVA)-specific immunogloblulin G1 (IgG1) but not IgG2a in OdDHL-treated OVA-immunized BALB/c mice compared to levels for controls. In vitro stimulation of lymphocytes from both Th1-biased C57Bl/6 and T-cell receptor transgenic mice and Th2-biased BALB/c mice in the presence of OdDHL demonstrated that OdDHL inhibits in vitro cytokine production in response to both mitogen and antigen, with gamma interferon (IFN-gamma) tending to be more inhibited than interleukin-4 (IL-4). In vitro mitogen or antigen restimulation of cells from mice treated with OdDHL in vivo shows effects on cytokine production which depend on the underlying immune bias of the mouse strain used, with a relative increase of IFN-gamma in Th1-biased C57Bl/6 mice and a relative increase of IL-4 in Th2-biased BALB/c mice. Thus, the mode of action of OdDHL on T-cell cytokine production is likely to be a relatively nonspecific one which accentuates an underlying immune response bias rather than one which specifically targets either Th1 or Th2 responses.     

Turned on by danger: activation of CD1d-restricted invariant natural killer T cells

CD1d-restricted invariant natural killer T (iNKT) cells bear characteristics of innate and adaptive lymphocytes, which allow them to bridge the two halves of the immune response and play roles in many disease settings. Recent work has characterized precisely how their activation is initiated and regulated. Novel antigens from important pathogens have been identified, as has an abundant self-antigen, β-glucopyranosylcaramide, capable of mediating an iNKT-cell response. Studies of the iNKT T-cell receptor (TCR)-antigen-CD1d complex show how docking between CD1d-antigen and iNKT TCR is highly conserved, and how small sequence differences in the TCR establish intrinsic variation in iNKT TCR affinity. The sequence of the TCR CDR3β loop determines iNKT TCR affinity for ligand-CD1d, independent of ligand identity. CD1d ligands can promote T helper type 1 (Th1) or Th2 biased cytokine responses, depending on the composition of their lipid tails. Ligands loaded into CD1d on the cell surface promote Th2 responses, whereas ligands with long hydrophobic tails are loaded endosomally and promote Th1 responses. This information is informing the design of synthetic iNKT-cell antigens. The iNKT cells may be activated by exogenous antigen, or by a combination of dendritic cell-derived interleukin-12 and iNKT TCR-self-antigen-CD1d engagement. The iNKT-cell activation is further modulated by recent foreign or self-antigen encounter. Activation of dendritic cells through pattern recognition receptors alters their antigen presentation and cytokine production, strongly influencing iNKT-cell activation. In a range of bacterial infections, dendritic cell-dependent innate activation of iNKT cells through interleukin-12 is the dominant influence on their activity.     

Host resistance and immune deviation in pigeon cytochrome c T-cell receptor transgenic mice infected with Toxoplasma gondii

Resistance to Toxoplasma gondii has been shown to be mediated by gamma interferon (IFN-gamma) produced by NK, CD4(+), and CD8(+) T cells. While studies of SCID mice have implicated NK cells as the source of the cytokine in acute infection, several lines of evidence suggest that IFN-gamma production by CD4(+) T lymphocytes also plays an important role in controlling early parasite growth. To evaluate whether this function is due to nonspecific as opposed to T-cell receptor (TCR)-dependent stimulation by the parasite, we have examined the resistance to T. gondii infection of pigeon cytochrome c transgenic (PCC-Tg) Rag-2(-/-) mice in which all CD4(+) T lymphocytes are unreactive with the protozoan. When inoculated with the ME49 strain, PCC-Tg animals exhibited only temporary control of acute infection and succumbed by day 17. Intracellular cytokine staining by flow cytometry revealed that, in contrast to infected nontransgenic controls, infected PCC-Tg animals failed to develop IFN-gamma-producing CD4(+) T cells. Moreover, the CD4(+) lymphocytes from these mice showed no evidence of activation as judged by lack of upregulated expression of CD44 or CD69. Nevertheless, when acutely infected transgenic mice were primed by PCC injection, the lymphokine responses measured after in vitro antigen restimulation displayed a strong Th1 bias which was shown to be dependent on endogenous interleukin 12 (IL-12). The above findings argue that, while T. gondii-induced IL-12 cannot trigger IFN-gamma production by CD4(+) T cells in the absence of TCR ligation, the pathogen is able to nonspecifically promote Th1 responses against nonparasite antigens, an effect that may explain the immunostimulatory properties of T. gondii infection.     

Instruction of naive CD4+ T-cell fate to T-bet expression and T helper 1 development: roles of T-cell receptor-mediated signals

Using T-cell receptor (TCR) transgenic mice, we demonstrate that TCR stimulation of naive CD4(+) T cells induces transient T-bet expression, interleukin (IL)-12 receptor beta2 up-regulation, and GATA-3 down-regulation, which leads to T helper (Th)1 differentiation even when the cells are stimulated with peptide-loaded I-A(b)-transfected Chinese hamster ovary cells in the absence of interferon-gamma (IFN-gamma) and IL-12. Sustained IFN-gamma and IL-12 stimulation augments naive T-cell differentiation into Th1 cells. Intriguingly, a significant Th1 response is observed even when T-bet(-/-) naive CD4(+) T cells are stimulated through TCR in the absence of IFN-gamma or IL-12. Stimulation of naive CD4(+) T cells in the absence of IFN-gamma or IL-12 with altered peptide ligand, whose avidity to the TCR is lower than that of original peptide, fails to up-regulate transient T-bet expression, sustains GATA-3 expression, and induces differentiation into Th2 cells. These results support the notion that direct interaction between TCR and peptide-loaded antigen-presenting cells, even in the absence of T-bet expression and costimulatory signals, primarily determine the fate of naive CD4(+) T cells to Th1 cells.     

Control of Borrelia burgdorferi-specific CD4+-T-cell effector function by interleukin-12- and T-cell receptor-induced p38 mitogen-activated protein kinase activity

Infection with Borrelia burgdorferi, the causative agent of Lyme disease, results in a Th1 response and proinflammatory cytokine production. Mice deficient for MKK3, an upstream activator of p38 mitogen-activated protein (MAP) kinase, develop a lower Th1 response and exhibit an impaired ability to produce proinflammatory cytokines upon infection with the spirochete. We investigated the contribution of p38 MAP kinase activity in gamma interferon (IFN-gamma) production in CD4+ T cells in response to specific antigen through T-cell receptor (TCR)- and interleukin-12 (IL-12)-mediated signals. The specific inhibition of p38 MAP kinase in T cells and the administration of a pharmacological inhibitor of the kinase during the course of infection with the spirochete resulted in reduced levels of IFN-gamma in the sera of infected mice. Our results also demonstrate that although p38 MAP kinase activity is not required for the differentiation of B. burgdorferi-specific CD4+ T cells, the production of IFN-gamma by Th1 effector cells is regulated by the kinase. Both TCR engagement and IL-12 induced the production of the Th1 cytokine through the activation of the p38 MAP kinase pathway. Thus, the inhibition of this pathway in vitro resulted in decreased levels of IFN-gamma during restimulation of B. burgdorferi-specific T cells in response to anti-CD3 and IL-12 stimulation. These results clarify the specific contribution of the p38 MAP kinase in the overall immune response to the spirochete and its role in the effector function of B. burgdorferi-specific T cells.     

Th1 cells induce and Th2 inhibit antigen-dependent IL-12 secretion by dendritic cells

Dendritic cells are the most relevant antigen-presenting cells (APC) for presentation of antigens administered in adjuvant to CD4⁺ T cells. Upon interaction with antigen-specific T cells, dendritic cells (DC) expressing appropriate peptide-MHC class II complexes secrete IL-12, a cytokine that drives Th1 cell development. To analyze the T cell-mediated regulation of IL-12 secretion by DC, we have examined their capacity to secrete IL-12 in response to stimulation by antigen-specific Th1 and Th2 DO11.10 TCR-transgenic cells. These cells do not differ either in TCR clonotype or CD40 ligand (CD40L) expression. Interaction with antigen-specific Th1, but not Th2 cells, induces IL-12 p40 and p75 secretion by DC. The induction of IL-12 production by Th1 cells does not depend on their IFN-γ secretion, but requires direct cell-cell contact mediated by peptide/MHC class II-TCR and CD40-CD40L interactions. Th2 cells not only fail to induce IL-12 secretion, but they inhibit its induction by Th1 cells. Unlike stimulation by Th1, inhibition of IL-12 production by Th2 cells is mediated by soluble molecules, as demonstrated by transwell cultures. Among Th2-derived cytokines, IL-10, but not IL-4 inhibit Th1-driven IL-12 secretion. IL-10 produced by Th2 cells appears to be solely responsible for the inhibition of Th1-induced IL-12 secretion, but it does not account for the failure of Th2 cells to induce IL-12 production by DC. Collectively, these results demonstrate that Th1 cells up-regulate IL-12 production by DC via IFN-γ-independent cognate interaction, whereas this is inhibited by Th2-derived IL-10. The inhibition of Th1-induced IL-12 production by Th2 cells with the same antigen specificity represents a novel mechanism driving the polarization of CD4⁺ T cell responses.     

CD4 Dependence of Activation Threshold and TCR Signalling in Mouse T Lymphocytes

The effect of CD4 expression on the activation threshold of mouse T lymphocytes has been analysed. To do this, the authors studied the response to antigen and other T cell receptor (TCR) ligands in a series of CD4^? mutants obtained from the SR.D10 clone. This non-tumour clone spontaneously arose from the Th2 clone D10.G4.1, and characteristically shows a low threshold for antigen activation as well as reactivity to syngeneic antigen presenting cells (APC). Although SR.D10 CD4^? mutant cells can be stimulated by antigen, they need higher antigen concentration or more APC than SR.D10 or CD4 transfectants to yield optimal antigen responses. Furthermore, CD4^? clones are not activated by syngeneic APC or by clonotypic antibodies. These effects do not correlate with changes in the expression of cell surface molecules implicated in antigen recognition, like TCR/CD3, CD2, LFA-1, or CD45, or with lower p56^lck or p59^fyn activity in the mutant cells. Since inhibition experiments using anti-CD4 antibodies have previously shown that activation of the CD4⁺ T cell clone D10.G4.1 by antigen or alloantigens is largely dependent on CD4, our results indicate that activation by antigen-plus self MHC may become CD4-independent if the activation threshold is lowered enough, e.g. in cells like SR.D10. Expression of CD4 further lowers the activation threshold of the cells, allowing the detection of low-affinity TCR reactivities like those directed at self MHC. Moreover, by using anti-TCR/CD3 antibodies, the authors have confirmed the importance of CD4-associated tyrosine kinase activity in early TCR/CD3 signalling in this Th2 cell line, as (1) upon TCR/CD3 ligation, tyrosine phosphorylation is detected only in those CD3 chains co-precipitating with CD4; and (2) CD4 expression is needed for efficient early tyrosine phosphorylation and detectable p56^lck-TCR co-precipitation.     

Interleukin-10 is expressed by bovine type 1 helper, type 2 helper, and unrestricted parasite-specific T-cell clones and inhibits proliferation of all three subsets in an accessory-cell-dependent manner.

Murine interleukin-10 (IL-10) is produced by type 2 helper (Th2) cells and selectively inhibits cytokine synthesis by type 1 helper (Th1) cells, whereas human IL-10 is produced by and inhibits proliferation and cytokine synthesis by both Th1 and Th2 subsets. This study reports that bovine IL-10 mRNA is expressed by Th0, Th1, and Th2 clones of bovine T cells specific for either Babesia bovis or Fasciola hepatica but not by two CD8+ T-cell clones. The antigen-induced proliferative responses of all three subsets of CD4+ cells were inhibited by human IL-10, and low levels (10 U/ml) of exogenous human IL-2 restored the suppressed response. However, proliferation of one Th1 clone was never inhibited but was enhanced by IL-10. Human IL-10 also inhibited the expression of gamma interferon and IL-4 mRNA in Th0 clones. In the absence of accessory cells (AC), the responses of Th clones to concanavalin A or IL-2 were not inhibited by IL-10, whereas antigen-specific responses of Th1 and Th2 cells were reduced when IL-10-pretreated macrophages were used as AC. Together, our results with bovine T cells support the concept that IL-10 primarily affects AC function and does not directly inhibit CD4+ T cells and demonstrate that the immunoregulatory effects of IL-10 are not selectively directed at Th1 populations, as they are in mice.     

Defective Th1 and Th2 cytokine synthesis in the T-T cell presentation model for lack of CD40/CD40 ligand interaction

In this study, T or NK cell clones used as antigen-presenting cells (T- or NK-APC) were shown to be significantly less efficient than professional APC in inducing Th1 and Th2 cytokines by antigen-specific T cell clones. This phenomenon was not related to a limited engagement of TCR by T-APC, since comparable thresholds of TCR down-regulation were shown when antigen was presented by either T-APC or professional APC. Rather, the stimulatory T-APC weakness was due to their inability, because they are CD40⁻, to provide the appropriate co-stimuli to responder T cells both indirectly via IL-12, and partially via direct CD40L triggering on T cells. Indeed, the simultaneous addition of IL-12 and reagents directly engaging CD40L on responder T cells restored T cell cytokine synthesis when antigen was presented by T-APC. In addition, either IL-12 production or blocking of T cell cytokine synthesis by anti-IL-12 p75 antibodies was evident only when professional APC were used in our antigen-specific system. The down-regulation of cytokine synthesis in the system of T-T cell presentation could represent a novel mechanism of immune regulation, which may intervene to switch off detrimental Th1- or Th2-mediated responses induced by antigen presentation among activated T cells infiltrating inflamed tissues.     

The nature and mechanisms of DN regulatory T-cell mediated suppression

Regulatory T cells have been reported to enhance survival of transplanted allografts. We have recently identified and cloned a novel CD3(+)CD4(-)CD8(-) (double negative, DN) regulatory T cell from mice that were given a single class I mismatched donor lymphocyte infusion and permanently accepted donor-specific skin allografts. When infused into na?ve syngeneic mice, these DN T cells prolonged the survival of class I mismatched donor skin allografts. Here we further characterize the nature and mechanism of DN T-cell mediated suppression. This present study reveals that DN T cells are able to specifically eliminate activated syngeneic CD8(+) T cells that share the same T cell receptor (TCR) specificity as DN T cells in vitro. Similarly, we found that, along with an increase of recipient DN T cells in the peripheral blood, anti-donor CD8(+) T cells were also eliminated in vivo following a donor lymphocyte infusion. We further demonstrate that DN T regulatory cells do not mediate suppression by competition for growth factors or antigen presenting cells (APC) nor by modulation of APC, but require cell contact with the activated target CD8(+) T cells. This contact can be mediated either by the TCR on CD8(+) T cells that recognize constitutively expressed or acquired MHC molecules on DN T cells, or by the TCR on DN T cells that recognize constitutively expressed MHC molecules on CD8(+) T cells. Together, these data extend our previous findings, and expand the conditions in which DN T cells can potentially be used to specifically suppress allogeneic immune responses.     

Modulation of experimental blood stage malaria through blockade of the B7/CD28 T-cell costimulatory pathway

Recent studies have implicated cytokines associated with CD4+ T lymphocytes of both T helper (Th)1 and Th2 subsets in resistance to experimental blood stage malaria. As the B7/CD28 costimulatory pathway has been shown to influence the differentiation of Th cell subsets, we investigated the contribution of the B7 molecules CD80 and CD86 to Th1/Th2 cytokine and immunoglobulin isotype profiles and to the development of a protective immune response to malaria in NIH mice infected with Plasmodium chabaudi. Effective blockade of CD86/CD28 interaction was demonstrated by elimination of interleukin (IL)-4 and up-regulation of interferon (IFN)-gamma responses by P. chabaudi-specific T cells and by reduction of P. chabaudi-specific immunoglobulin G1 (IgG1). The shift towards a Th1 cytokine pattern corresponded with efficient control of acute parasitaemia but an inability to resolve chronic infection. Moreover, combined CD80/CD86 blockade by using anti-CD80 and anti-CD86 monoclonal antibodies raised IFN-gamma production over that seen with CD86 blockade alone, with augmentation of this Th1-associated cytokine reducing levels of peak primary parasitaemia. These results demonstrate that IL-4 production by T cells in P. chabaudi-infected NIH mice is dependent upon CD86/CD28 interaction and that IL-4 and IFN-gamma contribute significantly, at different times of infection, to host resistance to blood stage malaria. In addition, combined CD80/CD86 blockade resulted in preferential expansion of IFN-gamma-producing T cells during P. chabaudi infection, suggesting that costimulatory pathways other than B7/CD28 may contribute to T-cell activation during continuous antigen stimulation. This study indicates a role for B7/CD28 costimulation in modulating the CD4+ T-cell response during malaria, and further suggests involvement of this pathway in other infectious and autoimmune diseases in which the Th cell immune response is also skewed.     

Glycosylation-dependent interaction of Jacalin with CD45 induces T lymphocyte activation and Th1/Th2 cytokine secretion

Jacalin, an alpha-O-glycoside of the disaccharide Thomsen-Friedenreich antigen (galactose beta1-3 N-acetylgalactosamine, T-antigen)-specific lectin from jackfruit seeds, has been shown to induce mitogenic responses and to block infection by HIV-1 in CD4+ T lymphocytes. The molecular mechanism underlying Jacalin-induced T cell activation has not been elucidated completely yet. In the present study, protein tyrosine phosphatase (PTPase) CD45 was isolated from a Jurkat T cell membrane fraction as a major receptor for Jacalin through affinity chromatography and mass spectrometry. CD45, which is highly glycosylated and expressed exclusively on the surface of lymphocytes, is a key regulator of lymphocyte signaling, playing a pivotal role in activation and development. We found that the lectin induced significant IL-2 production by a CD45-positive Jurkat T cell line (JE6.1) and primary T cells. However, this effect did not occur in a CD45-negative Jurkat T cell line (J45.01) and was blocked completely by a specific CD45 PTPase inhibitor in Jurkat T (JE6.1) and primary T cells. Furthermore, we also observed that Jacalin caused a marked increase in IL-2 secretion in response to TCR ligation and CD28 costimulation and contributed to Th1/Th2 cytokine production by activating CD45. Jacalin increased CD45 tyrosine phosphatase activity, which resulted in activation of the ERK1/2 and p38 MAPK cascades. Based on these findings, we propose a new, immunoregulatory model for Jacalin, wherein glycosylation-dependent interactions of Jacalin with CD45 on T cells elevate TCR-mediated signaling, which thereby up-regulate T cell activation thresholds and Th1/Th2 cytokine secretion.     

Antigen-induced regulation of T-cell motility,interaction with antigen-presenting cells and activation through endogenous thrombospondin-1 and its receptors

Antigen recognition reduces T-cell motility, and induces prolonged contact with antigen-presenting cells and activation through mechanisms that remain unclear. Here we show that the T-cell receptor (TCR) and CD28 regulate T-cell motility, contact with antigen-presenting cells and activation through endogenous thrombospondin-1 (TSP-1) and its receptors low-density lipoprotein receptor-related protein 1 (LRP1), calreticulin and CD47. Antigen stimulation induced a prominent up-regulation of TSP-1 expression, and transiently increased and subsequently decreased LRP1 expression whereas calreticulin was unaffected. This antigen-induced TSP-1/LRP1 response down-regulated a motogenic mechanism directed by LRP1-mediated processing of TSP-1 in cis within the same plasma membrane while promoting contact with antigen-presenting cells and activation through cis interaction of the C-terminal domain of TSP-1 with CD47 in response to N-terminal TSP-1 triggering by calreticulin. The antigen-induced TSP-1/LRP1 response maintained a reduced but significant motility level in activated cells. Blocking CD28 co-stimulation abrogated LRP1 and TSP-1 expression and motility. TCR/CD3 ligation alone enhanced TSP-1 expression whereas CD28 ligation alone enhanced LRP1 expression. Silencing of TSP-1 inhibited T-cell conjugation to antigen-presenting cells and T helper type 1 (Th1) and Th2 cytokine responses. The Th1 response enhanced motility and increased TSP-1 expression through interleukin-2, whereas the Th2 response weakened motility and reduced LRP1 expression through interleukin-4. Ligation of the TCR and CD28 therefore elicits a TSP-1/LRP1 response that stimulates prolonged contact with antigen-presenting cells and, although down-regulating motility, maintains a significant motility level to allow serial contacts and activation. Th1 and Th2 cytokine responses differentially regulate T-cell expression of TSP-1 and LRP1 and motility.     

Homotypic T-cell/T-cell interaction induces T-cell activation, proliferation, and differentiation

Activated CD4 T cells might induce T-cell activation from CD4 resting T cells in the absence of antigen presenting cells through interaction of activation-induced surface molecules (e.g., CD80, CD86, CD70, major histocompatibility complex class II) and their ligands constitutively expressed on resting T cells. Supporting this hypothesis, CD4 memory T cells proliferated in response to contact with activated T cells and expressed activation markers, such as CD25, CD30, and CD69. Analysis of their cytokine profile revealed differentiation of interleukin (IL)–10 and interferon-γ double-producing cells in response to contact with activated T helper (Th) 1 effector cells, and interleukin (IL)–4-producing cells in response to contact with activated Th2 effector cells. Whereas neutralization of interferon–γ or IL-4 during co-culture did not diminish the frequency of the arising cytokine-producing cells, separation of the responder cells from effector cells significantly decreased cytokine secretion. Specific blocking of particular receptor/ligand interactions denoted above could not prevent cytokine production induced by T-cell/T-cell interaction. However, blockade of all of the receptor/counterreceptor pairs significantly inhibited cytokine production, although not completely. Given the immunomodulatory capacity of IL-4 and IL-10, these findings might indicate a novel contact dependent negative feedback mechanism to control T-cell–driven immunity.     

Limiting dilution analysis of CD4 T-cell cytokine production in mice administered native versus polymerized ovalbumin: directed induction of T-helper type-1-like activation.

Polarized expression of T-helper type-1 (Th1)- or Th2-like patterns of cytokine production frequently correlates with disease outcome. Previously, we have described the long-lived reciprocal regulation of ovalbumin (OVA)-specific IgE (> 95% inhibition) and IgG2a (300-800-fold increased) production following administration of high MW OVA polymers (OVA-POL), in both de novo and ongoing OVA (alum)-induced responses. Here, limiting dilution analysis (LDA) was used to compare precursor frequencies of CD4 T cells producing interferon-gamma (IFN-gamma), interleukin-4 (IL-4) or IL-10 following OVA versus OVA-POL exposure in vivo. Adjuvants were not used, so as to circumvent their impact on measurement of precursor frequencies. We found that the two forms of antigen elicited T-cell activation of comparable intensity, as indicated by equivalent precursor frequencies of clonogenic antigen-specific CD4 T cells. However, they elicited qualitatively different cytokine responses. OVA-POL treatment led to 10-fold higher (mean of six independent LDA experiments) frequencies of IFN-gamma-producing cells, and a mean fivefold lower frequency of IL-10-producing cells, than was observed following in vivo administration of unmodified OVA. Thus, the high MW polymerized form of antigen acted to steer commitment of naive (for this antigen) CD4 T-cell activation from a situation in which IL-10 producers outnumbered IFN-gamma-producing cells by a factor of 4:1 (found in mice administered OVA), to one where IFN-gamma producers dominated by a factor of 11:1 (in mice given OVA-POL), i.e. a qualitative shift in the nature of the OVA-specific response induced from Th2-like to Th1-like. In vivo co-administration of anti-IFN-gamma monoclonal antibody (mAb) abolished the capacity of OVA-POL to preferentially elicit Th1-like dominance. Interestingly, although the ratios of IFN-gamma:IL-4 and IFN-gamma:IL-10 OVA-specific precursor frequencies were strongly increased following OVA-POL exposure (mean 18- and 47-fold higher), the frequency of IL-4-producing CD4 T cells did not differ significantly. The data suggest that this modified antigen promotes in vivo commitment of naive T cells towards a Th1-like response, with consequent inhibition of IgE and enhancement of IgG2a responses, not through direct effects on IL-4 production, but via decreased frequencies of IL-10 and increased frequencies of IFN-gamma-producing OVA-specific CD4 cells. Collectively, the data (1) demonstrate the ability to manipulate commitment of antigen-driven CD4 T-cell populations in naive mice to specific patterns of cytokine gene expression, and (2) provide in vivo evidence of the regulatory role played by IFN-gamma in limiting induction and/or expansion of IL-4- and IL-10-producing CD4 cells to protein allergens.     

The anthrax vaccine adsorbed vaccine generates protective antigen (PA)-Specific CD4+ T cells with a phenotype distinct from that of naive PA T cells

Cellular immune responses against protective antigen (PA) of Bacillus anthracis in subjects that received the anthrax vaccine adsorbed (AVA) vaccine were examined. Multiple CD4(+) T-cell epitopes within PA were identified by using tetramer-guided epitope mapping. PA-reactive CD4(+) T cells with a CD45RA(-) phenotype were also detected by direct ex vivo staining of peripheral blood mononuclear cells (PBMC) with PA-specific tetramers. Surprisingly, PA-specific T cells were also detected in PBMC of nonvaccinees after a single cycle of in vitro PA stimulation. However, PA-reactive CD4(+) T cells in nonvaccinees occurred at lower frequencies than those in vaccinees. The majority of PA-reactive T cells from nonvaccinees were CD45RA(+) and exhibited a Th0/Th1 cytokine profile. In contrast, phenotyping and cytokine profile analyses of PA-reactive CD4(+) T cells from vaccinees indicated that vaccination leads to commitment of PA-reactive T cells to a Th2 lineage, including generation of PA-specific, pre-Th2 central memory T cells. These results demonstrate that the current AVA vaccine is effective in skewing the development of PA CD4(+) T cells to the Th2 lineage. The data also demonstrated the feasibility of using class II tetramers to analyze CD4(+) cell responses and lineage development after vaccination.     

Multiparameter grouping delineates heterogeneous populations of human IL-17 and/or IL-22 T-cell producers that share antigen specificities with other T-cell subsets

The ontogenic relationship between pro-inflammatory populations of interleukin-17 (IL-17A)- and/or IL-22-producing T cells and other T-cell subsets is currently unclear in humans. To appreciate T helper cell-lineage commitment, we combined cytokine production profiles of in vitro expanded T-cell clones with T-cell receptor (TCR) clonotypic signatures. Moreover, ex vivo cytokine production profiles at the single-cell level were analyzed using an original approach based on the hierarchical cluster analysis of multiparametric flow cytometry data. These combined approaches enabled the delineation of distinct functional T-cell subsets, including Th1, Th2, Tr1, Th17 cells and a highly polyfunctional IL-22-producing T-cell population. Cluster analysis highlighted that the IL-22-producing T-cell population should be considered independently from the Th17 and Th1 subsets, although it was more closely related to the former. In parallel, we observed extensive TCRαβ sharing across all five subsets defined. The strategy described here allows the objective definition of cellular subsets and an unbiased insight into their similarities. Together, our results underscore the ontogenic plasticity of CD4(+) T-cell progenitors, which can adopt a differentiation profile irrespective of antigen specificity.     

Differential T‐cell receptor signals for T helper cell programming

Upon encounter with their cognate antigen, naive CD4 T cells become activated and are induced to differentiate into several possible T helper (Th) cell subsets. This differentiation depends on a number of factors including antigen‐presenting cells, cytokines and co‐stimulatory molecules. The strength of the T‐cell receptor (TCR) signal, related to the affinity of TCR for antigen and antigen dose, has emerged as a dominant factor in determining Th cell fate. Recent studies have revealed that TCR signals of high or low strength do not simply induce quantitatively different signals in the T cells, but rather qualitatively distinct pathways can be induced based on TCR signal strength. This review examines the recent literature in this area and highlights important new developments in our understanding of Th cell differentiation and TCR signal strength.